LOCUS pFH85 2868 bp DNA circular UNA 03-JUN-2019 DEFINITION Subcloning of protospacer into SpCas9 classic sgRNA backbone, overhang to pFH33 TaU3p. ACCESSION urn.local...1l-ajo6shz VERSION urn.local...1l-ajo6shz KEYWORDS . SOURCE ORGANISM . FEATURES Location/Qualifiers misc_feature 2..7 /Recognition_pattern="GGTCTC(1/5)" /annotation_group="GGTCTC" /label="BsaI" /note="Geneious type: restriction site" overhang 9..12 /created_by="hahnf" /label="AGCA to TaU3p pFH33" misc_feature complement(15..20) /Recognition_pattern="GAAGAC(2/6)" /annotation_group="GAAGAC" /label="BpiI" /note="Geneious type: restriction site" CDS 21..598 /created_by="hahnf" /label="lacZ" misc_feature 599..604 /Recognition_pattern="GAAGAC(2/6)" /annotation_group="GAAGAC" /label="BpiI" /note="Geneious type: restriction site" gRNA_Scaffold 607..682 /created_by="hahnf" /modified_by="hahnf" /label="SpCas9 classic sgRNA scaffold" overhang 607..610 /created_by="hahnf" /modified_by="hahnf" /label="GTTT" Stop_codon 683..691 /created_by="hahnf" /label="STOP" overhang 692..695 /created_by="hahnf" /modified_by="hahnf" /label="CGCT overhang to Level1 backbone" misc_feature complement(697..702) /Recognition_pattern="GGTCTC(1/5)" /annotation_group="GGTCTC" /label="BsaI" /note="Geneious type: restriction site" rep_origin complement(933..1547) /vntifkey=21 /X-Transferred_From="pAK-EL-01; pAK-EL-02; pAK-EL-03; pAK-EL-04; pAK-EL-05" /X-Transferred_Similarity="100.00%" /Transferred_From="pAK-EL-01, pAK-EL-02, pAK-EL-03, pAK-EL-04, pAK-EL-05, pAK007 in pUC57-Kan-mini, pAK008 in pUC57-Kan-mini, pAK009 in pUC57-Kan-mini, pAK010 in pUC57-Kan-mini, pAK011 in pUC57-Kan-mini, pAK012 in pUC57-Kan-mini" /Transferred_Similarity="100.00%" /label="rep(pMB1)" CDS complement(1672..2487) /vntifkey=21 /X-Transferred_From="pAK-EL-01; pAK-EL-02; pAK-EL-03; pAK-EL-04; pAK-EL-05" /X-Transferred_Similarity="100.00%" /Transferred_From="pAK-EL-01, pAK-EL-02, pAK-EL-03, pAK-EL-04, pAK-EL-05, pAK007 in pUC57-Kan-mini, pAK008 in pUC57-Kan-mini, pAK009 in pUC57-Kan-mini, pAK010 in pUC57-Kan-mini, pAK011 in pUC57-Kan-mini, pAK012 in pUC57-Kan-mini" /Transferred_Similarity="98.90%" /label="KanR" primer_bind 2558..2581 /Mismatches=0 /%GC="33.3" /Hairpin_Tm="None" /Self_Dimer_Tm="None" /Tm="56.0" /annotation_group="24555775085359fbfbe37b-b3de-415d-988b-2 576bc743911" /Sequence="GCAATGTAACATCAGAGATTTTGA" /created_by="primer3" /label="FH32" ORIGIN 1 gggtctcaag cattgtcttc gcagctggca cgacaggttt cccgactgga aagcgggcag 61 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt 121 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 181 cagctatgac catgattacg ccaagcttgc atgcctgcag gtcgactcta gaggatcccc 241 gggtaccgag ctcgaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 301 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg 361 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc 421 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc 481 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 541 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgacga 601 agacaagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga 661 aaaagtggca ccgagtcggt gctttttttt tcgcttgaga ccgaggctag gtggaggctc 721 agtgatgata agtctgcgat ggtggatgca tgtgtcatgg tcatagctgt ttcctgtgtg 781 aaattgttat ccgctcagag ggcacaatcc tattccgcgc tatccgacaa tctccaagac 841 attaggtgga gttcagttcg gcgtatggca tatgtcgctg gaaagaacat gtgagcaaaa 901 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 961 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 1021 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 1081 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 1141 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 1201 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 1261 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 1321 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 1381 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 1441 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 1501 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 1561 gggtctgacg ctctattcaa caaagccgcc gtcccgtcaa gtcagcgtaa atgggtaggg 1621 ggcttcaaat cgtccgctct gccagtgtta caaccaatta acaaattctg attagaaaaa 1681 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt 1741 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc 1801 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt 1861 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg 1921 tgagaatggc aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccattacg 1981 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc 2041 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg 2101 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa 2161 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt 2221 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac 2281 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg 2341 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg 2401 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctcgagca 2461 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga 2521 cagttttatt gttcatgatg atatattttt atcttgtgca atgtaacatc agagattttg 2581 agacacaacg tggctttccc ccgccgctct agaactagtg gatccaaata aaacgaaagg 2641 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcgcattat acgagacgtc 2701 caggttggga tacctgaaac aaaacccatc gtacggccaa ggaagtctcc aataactgtg 2761 atccaccaca agcgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtcatg 2821 cataatccgc acgcatctgg aataaggaag tgccattccg cctgacct //